Enzyme Mechanism - Examples
Can an enzyme's disulfide bond be redox active? A much more complicated redox enzyme is glutathione reductase, which uses NADPH to reduce oxidized glutathione in your cells. Glutathione is a 3 AA peptide (Glu-Cys-Gly) and two molecules are linked together by a disulfide bond in the oxidized form.
Figure 16. Reduction of glutathione by NADPH catalyzed by glutathione reductase.
Figure 17. Structural Model of Glutathione Reductase showing the domains for binding the internal flavin molecule (FAD) and the reducing substrate, NADPH, and the interface domain for joining the two subunits. The active site where glutathione is bound is between the two subunits and it is here where the redox active disulfide of the enzyme is located.
To achieve reduction of GSSG to GSH glutathione reductase must split the two electrons provided by NADPH and give them one at a time to each of the sulfurs of the GSSG. To do this, glutathione reductase contains FAD and it also uses a redox active enzyme disulfide. FAD differs from NADH/NADPH in that it can accept 2 electrons and pass them one at a time.
Figure 18. Structure of flavin adenine dinucleotide (FAD) and how it can be reduced in 2 one-electron transfers. It can also be oxidized in two one-electron transfers, which allows it to split the electrons of NADPH to use for reduction of glutathione in two steps each involving the transfer of 1 electron.
Glutathione reductase acts in two steps:
First, NADPH reduces the enzyme and second the reduces GSSG to form 2 GSH.
Figure 19. The steps in Glutathione Reductase catalytic mechanisms showing the reduction of the enzyme by NADPH (Tyr 197 is involved in binding NADPH) where the FAD is converted to FADH2. Next reduced FAD reacts with the internal disulfide of the enzyme where one -SH group is stabilized by interaction with the FAD while the other is stabilized by His 467 in combination with Glu 272. Next glutathione (G-S-S-G) reacts with the -SH group bound to the His-467 leading to one molecule of GSH (reduced glutathione being released) and a mixed disulfide formed between the second glutathione and the Enzyme's thiol (G-S-S-Enzyme). Finally, the enzyme's -SH-FAD complex attacks the G-S-S-Enzyme to displace the second GSH (which is released) and regenerating the enzyme's disulfide (E-S-S-E).
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu